Erratum in

J Neuropathol Exp Neurol. 2014 Feb;73(2):190.

Abstract

Hereditary spastic paraplegias (HSPs) are characterized by progressive spasticity and weakness in the lower extremities that result from length-dependent central to peripheral axonal degeneration. Mutations in the non-imprinted Prader-Willi/Angelman syndrome locus 1 (NIPA1) transmembrane protein cause an autosomal dominant form of HSP (SPG6). Here, we report that transgenic (Tg) rats expressing a human NIPA1/SPG6 mutation in neurons (Thy1.2-hNIPA1) show marked early onset behavioral and electrophysiologic abnormalities. Detailed morphologic analyses reveal unique histopathologic findings, including the accumulation of tubulovesicular organelles with endosomal features that start at axonal and dendritic terminals, followed by multifocal vacuolar degeneration in both the CNS and peripheral nerves. In addition, the NIPA1 mutation in the spinal cord from older Tg rats results in an increase in bone morphogenetic protein type II receptor expression, suggesting that its degradation is impaired. This Thy1.2-hNIPA1 Tg rat model may serve as a valuable tool for understanding endosomal trafficking in the pathogenesis of a subgroup of HSP with an abnormal interaction with bone morphogenetic protein type II receptor, as well as for developing potential therapeutic strategies for diseases with axonal degeneration and similar pathogenetic mechanisms.

Thy1.2-hNIPA1G106R transgenic (Tg) rat. (A) The transgene construct was created using the mouse Thy1.2 promoter and human NIPA1G106R mutant cDNA. (B) RT-PCR of the transgene, human non-imprinted Prader-Willi/Angelman syndrome locus 1 (NIPA1) (first row), endogenous rat NIPA1 (third row), and glyceraldehyde-3-phosphate dehydrogenase ([GAPDH] fifth row) in major organs from Thy1.2-hNIPAG106R rat at 4 weeks of age. Transgene expression was detected specifically in the brain and spinal cord compared with ubiquitous expression of endogenous NIPA1. NC, negative control (absence of RNA template); RT, reverse transcriptase. (C) Quantitative PCR of endogenous NIPA1 mRNA expression in the brain and spinal cord between age-matched (4 weeks) wild-type (WT) and Thy1.2-hNIPAG106R rats (n = 3 in each group). WT NIPA1 expression standardized by GAPDH was set as 1. There was no apparent difference in endogenous NIPA1 expression between WT and Thy1.2-hNIPAG106R Tg rats. NS, not significant. (D) Age-matched young (4 weeks) and old (33 weeks) WT and Thy1.2-hNIPA1G106R Tg rats (n = 3 in each group) were compared for NIPA1 protein expression (standardized by β-actin is set as 1). There was no apparent difference detected in the amount of NIPA1 between WT and Thy1.2-hNIPA1G106R Tg rats at different ages.

Thy1.2-hNIPA1G106R transgenic (Tg) rat spinal cord protein. (A) Immunoblot of spinal cord protein extracts from age-matched (33 weeks) wild-type (WT) and Tg rats with anti–non-imprinted Prader-Willi/Angelman syndrome locus 1 (NIPA1), bone morphogenetic protein type II receptor (BMPRII), and β-actin (loading control) antibodies. NIPA1 was detected as an approximately 34-kDa and an approximately 40-kDa band. (B) Age-matched young (4 weeks) and old (33 weeks) WT and Tg rats (n = 3 in each group) were compared for BMPRII expression. WT BMPRII expression standardized by β-actin was set as 1. BMPRII was significantly increased (∼2-fold) in the spinal cords only from the older Tg rats versus age-matched WT rats (* p < 0.01). No apparent difference in the amount of BMPRII was detected in brains from either young or old Tg rats. (C) Immunoblot of nuclear fraction protein extracts of primary cortical nerve culture derived from WT and Tg rats with anti–phosphorylated Smad (p-Smad) and –β-actin antibodies, in response to either bone morphogenetic protein 9 (BMP9) (BMP[+]) or vehicle. Time from the application of either BMP9 or vehicle is shown (in minutes). Increased p-Smad was seen only in the extracts of nerve cells exposed to BMP9, whereas no detectable response was seen in vehicle controls. (D) Time course of p-Smad activation in response to BMP9 onto primary rat cortical neuron culture. Response of p-Smad at 120-minute exposure to BMP9 ligand in WT nerve culture, standardized by β-actin was set as 1. No apparent difference in the response of p-Smad to BMP ligand was seen between WT and Tg rat primary cortical nerve culture (data set was triplicated; p = 0.42).

Schematic illustration depicts the evolution of neurodegeneration in autosomal dominant hereditary spastic paraplegia (SPG6-HSP). (A) Normal spinal motor neuron. At the early stages of the disease process, the tubulovesicular membranous structures with endosomal identity accumulated at the axonal and dendritic nerve endings, along the distal axons and in the neuronal cell bodies. This accumulation was likely caused by an impaired transport and/or impaired recycling of endosomes. (B–D) A rapidly evolving multifocal intraneuronal accumulation of these aggregates sequestered by a double limiting membrane for autophagy was observed within axons (B) and dendrites (C). Multifocal vacuolar degeneration then occurred in distal axons and dendritic extensions extending pari passu in a centripetal direction with concomitant cell body involvement (C, D).